The long-term objective of the proposed research is to understand how information from the three signaling centers of the developing limb bud is integrated in space and developmental time to produce the patterned amniote limb. In order to do this (i) components of the signaling center pathways that are presently unknown must be discovered, and (ii) a means of integrating the monotonic signaling center effectors determined. In the past grant period, we defined the molecular biology of a limbless mutant. We propose the hypothesis that the chicken limbless mutant is an important tool for using differential cloning techniques to study these two significant problems in embryonic pattern formation. Subtraction of cDNAs common to limbless and wild-type limb buds yielded a cDNA library enriched for signaling pathway components. Preliminary results give strong support to the validity of the hypothesis. Following up on the preliminary data we propose the following three specific aims. 1) Continue to screen the signaling center pathway-enriched cDNA library to isolate novel cDNAs that are expressed in spatially and temporally restricted patterns and to analyze them for their role in limb pattern formation by epistatic analysis to known limb pattern genes and misexpression using viral techniques. 2) Determine the role in limb development of a chicken (c) homolog of the C. elegans heterochronic gene Lin-41 which we propose as a timing gene along the proximal-distal axis of the developing limb; cLin-41 was isolated in the initial screen. 3) Determine the role of a Wnt7a-FGF responsive gene isolated in the initial screen for its function in the dorsal-ventral signaling pathway downstream of known components in this pathway; or, alternatively, in a parallel pathway whose function is proposed to maintain Sonic hedgehog signaling in the limb bud. The proposed research will produce new information on cell signaling in embryonic development. By defining the role of a putative chicken heterochronic gene in limb development we will be able to better understand how developmental timing along the limb proximal-distal axis is controlled. This could lead to insights at the molecular level of malformations such as phocomelia. Similarly, by defining the role of the unique Wnt7a-FGF responsive gene we will significantly advance understanding of how this complex pathway functions in limb development.